ANTONIO ANDERSON DE JESUS RODRIGUES et al. 343 TECHNICAL ARTICLE Photoperiod and growth regulators on in vitro shoot induction in Heliconia latispatha(1) ANTONIO ANDERSON DE JESUS RODRIGUES(2), EDER DE OLIVEIRA SANTOS(3) and ANA CRISTINA PORTUGAL PINTO DE CARVALHO(4)* ABSTRACT Considering the growing economic importance of tropical flowers and the advantages of techniques applied to the in vitro cultivation of these plants, it is necessary to carry out studies to evaluate growth in species such as Heliconia latispatha. The aim of this study therefore, was to evaluate in vitro shoot induction for different concentrations of BAP and NAA and as a function of the photoperiod. Explants from zygotic embryos were inoculated in MS medium containing different concentrations of BAP (0.0, 0.5, 1.0, 1.5, 2.0 or 2.5 mg L-1), with the cultures kept in a growth room at a temperature of 24.0 ± 2.0° C, under a photoperiod of 12 and 16 hours of light and a light intensity of 30 μmol m-2 s-1. At 21, 28, 35, 42 and 49 days after inoculation, the number of shoots per explant was evaluated. The treatment at the BAP concentration that gave the best multiplication rate (2.5 mg L-1) was set, and was tested in a further trial with different concentrations of NAA (0.0, 0.2, 0.4, 0 6, 0.8 or 1.0 mg L-1) under the same conditions as the previous experiment. The experimental designs were completely randomised, with five replications, and analysed in a 6 x 2 factorial. The data were submitted to analysis of variance and regression. No significant differences were seen in relation to the photoperiod or its interaction with the cytokinin and auxin under test. Multiplication was greater in the presence of 2.5 mg L-1 BAP, which gave a rate of 1.25 shoots/explant at 49 days of in vitro culture. The association of this BAP dosage with 1.0 mg L-1 NAA was even more efficient, producing 1.83 shoots per explant at 30 days of growth. The use of BAP together with NAA is beneficial to the induction of shoots in H. latispatha. Keywords: Heliconiaceae, 6-benzylaminopurine, naphthaleneacetic acid, tissue culture. RESUMO Indução de brotações in vitro de Heliconia latispatha em função do fotoperíodo e reguladores de crescimento Considerando a crescente importância econômica das flores tropicais e as vantagens das técnicas aplicadas ao cultivo in vitro para estas plantas, torna-se necessária a realização de estudos que avaliem a propagação de espécies como a Heliconia latispatha. Diante do exposto, o objetivo do trabalho foi avaliar a indução de brotações in vitro em função de diferentes concentrações de BAP e ANA em função de fotoperíodos. Os explantes, oriundos de embriões zigóticos, foram inoculados em meio MS contendo diferentes concentrações de BAP (0,0; 0,5; 1,0; 1,5; 2,0 ou 2,5 mg L-1), mantendo-se as culturas em sala de crescimento com temperatura de 24,0 ± 2,0 ºC sob fotoperíodo de 12 e 16 horas de luz e intensidade luminosa de 30 μmol m-2 s-1. Aos 21, 28, 35, 42 e 49 dias após a inoculação, foi avaliado o número de brotos obtidos por explante. O tratamento com a concentração de BAP que proporcionou melhor taxa de multiplicação (2,5 mg L-1) foi fixado e, em outro ensaio foi testado com diferentes concentrações de ANA (0,0; 0,2; 0,4; 0,6; 0,8 ou 1,0 mg L-1) nas mesmas condições do experimento anterior. O delineamento experimental dos experimentos foi o inteiramente casualizado, com cinco repetições, analisado em esquema fatorial 6 x 2. Os dados foram submetidos à análise de variância e à regressão. Não foram observadas diferenças significativas com relação ao fotoperíodo nem sua interação com a citocinina ou auxina testada. A taxa de multiplicação foi maior na presença de 2,5 mg L-1 de BAP, que proporcionou uma taxa de multiplicação de 1,25 brotos/explante aos 49 dias de cultivo in vitro. A associação dessa dosagem de BAP com 1,0 mg L-1 de ANA foi ainda mais eficiente produzindo 1,83 brotos por explante em 30 dias de cultivo. O uso associado de BAP e ANA é benéfico para a indução in vitro de brotações de H. latispatha. Palavras-chave: Heliconiaceae, 6-benzilaminopurina, ácido naftaleno acético, cultura de tecidos. (1) Received in 17/08/2016 and accepted in 18/11/2016 (2) Universidade Federal do Ceará (UFC), Departamento de Fitotecnia, Fortaleza-CE, Brazil. (3) Universidade Federal do Ceará (UFC), Departamento de Ciências do Solo, Fortaleza-CE, Brazil. (4) Embrapa Agroindústria Tropical (EMBRAPA CNPAT), Laboratório de Cultura de Tecidos Vegetais, Fortaleza-CE, Brazil. *Corresponding author: [email protected] CAMPINAS-SP | V. 22, No. 3, 2016, p. 343-349 344 PHOTOPERIOD AND GROWTH REGULATORS ON IN VITRO SHOOT INDUCTION IN HELICONIA LATISPATHA 1. INTRODUCTION date not been reported in the literature, it becomes necessary to establish responsive micropropagation protocols for the Over the past few years, the Brazilian market for flowers production of plantlets of high phytosanitary and genetic and ornamental plants has been growing dynamically. In quality on a large scale, in order to meet the demands of 2013, floriculture in Brazil had a turnover of approximately commercial production. The aim of this study therefore was BRL 1.49 billion, an increase of 57.56% (JUNQUEIRA to evaluate the influence of the growth regulators BAP and and PEETZ, 2014). NAA and of photoperiod on the in vitro shoot induction of Considered profitable, the cultivation of tropical Heliconia latispatha Bentham cv. Orange Gyro. flowers has been on the increase and is now regarded as a viable alternative in small rural areas (COELHO et al., 2. MATERIAL AND METHODS 2016). Heliconia stands out among tropical flowers due to its high consumer acceptance, a result of the shape and The explants were obtained from cultures previously intense colouring and exoticism of the inflorescence, and to established in vitro from zygotic embryos and multiplied its high post-harvest durability (LIMA et al., 2016). by successive subcultures, under a photoperiod of 12 and Castro et al. (2007), evaluating 30 genotypes of 16 hours, in a basic MS culture medium (MURASHIGE heliconia for suitability as cut flowers, included the species and SKOOG, 1962) supplemented with 30 g L-1 sucrose Heliconia lathispatha among those classified as moderately and solidified with 5.5 g L-1 agar. suitable. Among its features, the inflorescence of the The shoots used as explants had an average height of species was classified as having a light orange coloration, 2.5 cm, a diameter of 5.5 mm, from 3 to 4 leaves and were with flowering spread over the summer months (January to without roots. The pH of the culture medium was adjusted March) and the beginning of autumn (April), and flowering to 5.8 and the medium autoclaved at 121°C and 1 atm for intervals of 90 to 120 days. It should be noted that, of the 20 minutes. The culture medium was supplemented with species of heliconia classified as moderately suitable for six concentrations of BAP (0.0, 0.5, 1.0, 1.5, 2.0 or 2.5 mg cut flowers, Heliconia latispatha is still little cultivated or L-1), to give a total of 12 treatments. traded in the domestic market. Glass jars were used, 10 cm in height and 6.3 cm in In vitro techniques of propagation of flowers and internal diameter, having a volume of 220 mL and with a ornamental plants are being increasingly used commercially, polypropylene screw cap; these contained 30 mL of culture as the floriculture sector demands a great number of uniform medium. The explants were inoculated into the nutrient plants of high genetic and phytosanitary quality throughout medium under aseptic conditions in a laminar flow cabinet. the year (CARVALHO et al., 2013). The experimental design was completely randomised In the tissue culture laboratory, luminosity in the growth with five replications per treatment, and analysed in a 2 x 6 rooms is an important factor, as this has an influence on factorial (two photoperiods x six concentrations of BAP), the proper development of the plants in vitro (BARRUETO giving a total of 60 plots. Each replication consisted of CID and TEIXEIRA, 2010). In micropropagation, the most one jar containing four explants. The cultures were kept widely used photoperiods are 12 hours of light / 12 hours of in two growth rooms, at photoperiods of 12 and 16 hours darkness or 16 hours light / 8 hours of darkness (RIBEIRO according to treatment, both rooms at a temperature of 24 ± et al., 2009; SILVA et al., 2012; GONZALEZ and CUEVA, 2°C and a light intensity of 30 μmol m-2 s-1. 2014; SANTOS et al., 2015; SANTOS et al., 2016). At 21, 28, 35, 42 and 49 days after inoculation of the In most of the studies carried out with different species explants in vitro, an evaluation was made of the number of of heliconia, the photoperiod used was 16 hours (NATHAN shoots per explant. et al., 1992; DINIZ et al., 2004; RODRIGUES et al., 2006; The best concentration of BAP was 2.5 mg L-1, which GUZMAN et al., 2009; ALARCÓN et al., 2011), however was then combined with different concentrations of the authors did not evaluate the effect of photoperiod on naphthaleneacetic acid (NAA) (0.0, 0.2, 0.4, 0.6, 0.8 or the in vitro micropropagation of the species (BRAGA et 1.0 mg L-1) at two photoperiods (12 and 16 hours).